We propose to use photochemical crosslinking techniques to investigate the structure of (a) the binding sites of 30S ribosomal subunit proteins S4, S7, S8, S15, and S20 on the E. coli 16S ribosomal RNA and of (b) the P and A transfer RNA binding sites of E. coli ribosomes. Radioactive aryl azide groups will be attached to specific cysteine or lysine residues of proteins S4, S7, S8, S15 and S20. The modified proteins will be allowed to form specific complexes with the S16 RNA. The azide groups on the proteins will then be decomposed photochemically to nitrenes which should insert into nearby regions of the rRNA. The sites of nitrene insertion in the rRNA will be determined by nucleic acid sequencing methods. The results will provide information as to the specific segments of the S16 RNA and of the ribosomal proteins that neighbor one another in the ribonucleoprotein complex. An aryl azide group has been attached to the 4-thiouracil residue at the base of the DHU stem in E. coli tRNA1val. Irradiation of the azide group of the derivatized tRNA while the latter is noncovalently bound to the ribosomal P site results in crosslinking between the 4-thiouracil and S16 RNA of the S30 subunit. We propose to determine which sequences of the rRNA are involved in this crosslink. Aryl azide groups will be placed at other specific regions of the tRNA molecule including the extra arm, the anticodon loop and the anticodon itself. These derivatized tRNAs will also be irradiated while bound to the ribosomal P site, and the components of the ribosome which become attached to the tRNA as a result of nitrene insertion will be determined. Similar experiments will be carried out with tRNAs bound to the ribosomal A site. In addition to defining the molecular architecture of the P and A binding sites of the ribosome, these studies are expected to provide a means for determining the spatial arrangement of the S16 RNA in the S30 subunit.